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1.
Hum Mol Genet ; 23(7): 1856-68, 2014 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-24234652

RESUMEN

Congenital myasthenic syndromes (CMS) are heterogeneous disorders in which the safety margin of neuromuscular transmission is compromised by one or more specific mechanisms. Using Sanger and exome sequencing in a CMS patient, we identified two heteroallelic mutations, p.Glu1233Lys and p.Arg1277His, in LRP4 coding for the postsynaptic low-density lipoprotein receptor-related protein 4. LRP4, expressed on the surface of the postsynaptic membrane of the neuromuscular junction, is a receptor for neurally secreted agrin, and LRP4 bound by agrin activates MuSK. Activated MuSK in concert with Dok-7 stimulates rapsyn to concentrate and anchor AChR on the postsynaptic membrane and interacts with other proteins implicated in the assembly and maintenance of the neuromuscular junction. LRP4 also functions as an inhibitor of Wnt/beta-catenin signaling. The identified mutations in LRP4 are located at the edge of its 3rd beta-propeller domain and decrease binding affinity of LRP4 for both MuSK and agrin. Mutations in the LRP4 3rd beta-propeller domain were previously reported to impair Wnt signaling and cause bone diseases including Cenani-Lenz syndactyly syndrome and sclerosteosis-2. By analyzing naturally occurring and artificially introduced mutations in the LRP4 3rd beta-propeller domain, we show that the edge of the domain regulates the MuSK signaling whereas its central cavity governs Wnt signaling. We conclude that LRP4 is a new CMS disease gene and that the 3rd beta propeller domain of LRP4 mediates the two signaling pathways in a position-specific manner.


Asunto(s)
Agrina/metabolismo , Proteínas Relacionadas con Receptor de LDL/genética , Síndromes Miasténicos Congénitos/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Colinérgicos/metabolismo , Adolescente , Animales , Secuencia de Bases , Células COS , Línea Celular , Chlorocebus aethiops , Agonistas Colinérgicos/uso terapéutico , Inhibidores de la Colinesterasa/uso terapéutico , Edrofonio/uso terapéutico , Activación Enzimática/genética , Femenino , Células HEK293 , Humanos , Ratones , Proteínas Musculares/metabolismo , Mutación , Unión Neuromuscular/metabolismo , Estructura Terciaria de Proteína , Bromuro de Piridostigmina/uso terapéutico , Análisis de Secuencia de ADN , Proteínas Wnt/antagonistas & inhibidores , Vía de Señalización Wnt/genética , beta Catenina/antagonistas & inhibidores
2.
Proc Natl Acad Sci U S A ; 110(12): 4714-9, 2013 Mar 19.
Artículo en Inglés | MEDLINE | ID: mdl-23471986

RESUMEN

Although endogenous ligands for Toll-like receptor (TLR)4-myeloid differentiation factor 2 (MD2) have not been well-understood, we here report that a globo-series glycosphingolipid, globotetraosylceramide (Gb4), attenuates the toxicity of lipopolysaccharides (LPSs) by binding to TLR4-MD-2. Because α1,4-galactosyltransferase (A4galt)-deficient mice lacking globo-series glycosphingolipids showed higher sensitivity to LPS than wild-type mice, we examined mechanisms by which globo-series glycosphingolipids attenuate LPS toxicity. Cultured endothelial cells lacking A4galt showed higher expression of LPS-inducible genes upon LPS treatment. In turn, introduction of A4galt cDNA resulted in the neo expression of Gb4, leading to the reduced expression of LPS-inducible genes. Exogenous Gb4 induced similar effects. As a mechanism for the suppressive effects of Gb4 on LPS signals, specific binding of Gb4 to the LPS receptor TLR4-MD-2 was demonstrated by coprecipitation of Gb4 with recombinant MD-2 and by native PAGE. A docking model also supported these data. Taken together with colocalization of TLR4-MD-2 with Gb4 in lipid rafts after LPS stimulation, it was suggested that Gb4 competes with LPS for binding to TLR4-MD-2. Finally, administration of Gb4 significantly protected mice from LPS-elicited mortality. These results suggest that Gb4 is an endogenous ligand for TLR4-MD-2 and is capable of attenuating LPS toxicity, indicating the possibility for its therapeutic application in endotoxin shock.


Asunto(s)
Globósidos/inmunología , Antígeno 96 de los Linfocitos/inmunología , Complejos Multiproteicos/inmunología , Receptor Toll-Like 4/inmunología , Animales , Galactosiltransferasas/genética , Galactosiltransferasas/inmunología , Galactosiltransferasas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Globósidos/genética , Globósidos/metabolismo , Lipopolisacáridos/toxicidad , Antígeno 96 de los Linfocitos/genética , Antígeno 96 de los Linfocitos/metabolismo , Microdominios de Membrana/genética , Microdominios de Membrana/inmunología , Microdominios de Membrana/metabolismo , Ratones , Ratones Mutantes , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Unión Proteica , Choque Séptico/inducido químicamente , Choque Séptico/genética , Choque Séptico/inmunología , Choque Séptico/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo
3.
Am J Physiol Gastrointest Liver Physiol ; 309(4): G260-9, 2015 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-26089335

RESUMEN

Although cystic fibrosis is rare in Japanese, measurement of sweat Cl(-) has suggested mild dysfunction of cystic fibrosis transmembrane conductance regulator (CFTR) in some patients with chronic pancreatitis. In the present study, we have investigated the association of CFTR variants and chronic pancreatitis in Japanese and the functional characteristics of a Japanese- and pancreatitis-specific CFTR variant, L1156F. Seventy patients with alcoholic chronic pancreatitis, 18 patients with idiopathic chronic pancreatitis, and 180 normal subjects participated. All exons and their boundaries and promoter region of the CFTR gene were sequenced. Human embryonic kidney-293 cells were transfected with three CFTR variants (M470V, L1156F, and M470V+L1156F), and the protein expression was examined. Xenopus laevis oocytes were injected with the CFTR variants, and bicarbonate (HCO3 (-)) transport activity was examined. CFPAC-1 cells were transfected with the CFTR variants and Cl(-)/HCO3 (-) exchange activity was examined. Six variants (E217G, I556V, M470V, L1156F, Q1352H, and R1453W) were identified in the coding region of the CFTR gene. Cystic fibrosis-causing mutations were not found. The allele frequencies of L1156F and Q1352H in alcoholic chronic pancreatitis (5.0 and 7.9%) were significantly (P < 0.01) higher than those in normal subjects (0.6 and 1.9%). L1156F was linked with a worldwide CFTR variant, M470V. Combination of M470V and L1156F significantly reduced CFTR expression to ∼60%, impaired CFTR-mediated HCO3 (-)/Cl(-) transport activity to 50-60%, and impaired CFTR-coupled Cl(-)/HCO3 (-) exchange activity to 20-30%. The data suggest that the Japanese-specific CFTR variant L1156F causes mild dysfunction of CFTR and increases the risk of alcoholic chronic pancreatitis in Japanese.


Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Mutación Missense , Pancreatitis Alcohólica/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Bicarbonatos/metabolismo , Estudios de Casos y Controles , Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Exones , Femenino , Frecuencia de los Genes , Células HEK293 , Humanos , Transporte Iónico , Japón , Masculino , Persona de Mediana Edad , Xenopus
4.
J Org Chem ; 80(17): 8561-70, 2015 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-26258850

RESUMEN

Here, we report the synthesis of a hybridization probe for detection of RNA and DNA based on photoinduced electron transfer (PeT). We designed and synthesized an oligonucleotide containing an adenosine analogue with a 9-(N,N-dimethylaminomethyl)anthracenyl moiety at its 6-position via an ethynylene linker as the hybridization probe. When the probe was hybridized with a complementary RNA or DNA, the fluorescence intensity increased 3-fold or 4.5-fold, respectively, compared to the single-stranded state.


Asunto(s)
Adenosina/análogos & derivados , Adenosina/química , Antracenos/química , Sondas de ADN/química , ADN/química , Oligonucleótidos/síntesis química , ARN/química , Transporte de Electrón , Electrones , Fluorescencia , Oligonucleótidos/química
5.
J Neurosci ; 27(28): 7604-15, 2007 Jul 11.
Artículo en Inglés | MEDLINE | ID: mdl-17626222

RESUMEN

Drug addiction places an enormous burden on society through its repercussions on crime rate and healthcare. Repeated exposure to drugs of abuse causes cellular adaptations in specific neuronal populations that ultimately can lead to a state of addiction. In the present study, we have identified a novel molecule "shati" from the nucleus accumbens (NAc) of mice treated with methamphetamine (METH) using the PCR-select complementary DNA subtraction method. Moreover, we investigated whether shati is involved in METH-induced hyperlocomotion, sensitization, and conditioned place preference (CPP). METH induced expression of shati mRNA dose dependently via dopamine (DA) receptors. We prepared antibodies against shati and, using them, found shati to be expressed in neuronal cells of the mouse brain. Treatment with the shati antisense oligonucleotide (shati-AS), which significantly inhibited the expression of shati mRNA, enhanced the acute METH response, METH-induced behavioral sensitization, and CPP. Blockage of shati mRNA by shati-AS potentiated the METH-induced increase of DA overflow in the NAc and the METH-induced decrease in synaptosomal and vesicular DA uptake in the midbrain. These results suggest that a novel molecule shati is involved in the development of METH-induced hyperlocomotion, sensitization, and CPP. The functional roles of shati in METH-regulated behavioral alternations are likely to be mediated by its inhibitory effects on the METH-induced increase of DA overflow in the NAc and the METH-induced decrease in DA uptake in the midbrain.


Asunto(s)
Acetiltransferasas/metabolismo , Conducta Animal/efectos de los fármacos , Condicionamiento Psicológico/fisiología , Discinesia Inducida por Medicamentos , Metanfetamina/metabolismo , Metanfetamina/farmacología , Acetiltransferasas/genética , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Dopamina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Distribución Tisular
6.
J Neurosci ; 26(12): 3335-44, 2006 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-16554484

RESUMEN

Glial cell line-derived neurotrophic factor (GDNF) is an important neurotrophic factor that has therapeutic implications for neurodegenerative disorders. We previously showed that leucine-isoleucine (Leu-Ile), an analog of a dipeptide-like structure of FK506 (tacrolimus), induces GDNF expression both in vivo and in vitro. In this investigation, we sought to clarify the cellular mechanisms underlying the GDNF-inducing effect of this dipeptide. Leu-Ile transport was investigated using fluorescein isothiocyanate-Leu-Ile in cultured neurons, and the results showed the transmembrane mobility of this dipeptide. By liquid chromatography-mass spectrometry and quartz crystal microbalance assay, we identified heat shock cognate protein 70 as a protein binding specifically to Leu-Ile, and molecular modeling showed that the ATPase domain is the predicted binding site. Leu-Ile stimulated Akt phosphorylation, which was attenuated significantly by heat shock protein 90 (Hsp90) inhibitor geldanamycin (GA). Moreover, enhanced interaction between phosphorylated Akt and Hsp90 was detected by immunoprecipitation. Leu-Ile elicited an increase in cAMP response element binding protein (CREB) phosphorylation, which was inhibited by GA, indicating that CREB is a downstream target of Hsp90/Akt signaling. Leu-Ile elevated the levels of GDNF mRNA and protein expression, whereas inhibition of CREB blocked such effects. Leu-Ile promoted the binding activity of phosphorylated CREB with cAMP response element. These findings show that CREB plays a key role in transcriptional regulation of GDNF expression induced by Leu-Ile. In conclusion, Leu-Ile activates Hsp90/Akt/CREB signaling, which contributes to the upregulation of GDNF expression. It may represent a novel lead compound for the treatment of dopaminergic neurons or motoneuron diseases.


Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Dipéptidos/farmacología , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Tacrolimus/análogos & derivados , Animales , Sitios de Unión/fisiología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/efectos de los fármacos , Dipéptidos/química , Inhibidores Enzimáticos/farmacología , Factor Neurotrófico Derivado de la Línea Celular Glial/efectos de los fármacos , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Proteínas del Choque Térmico HSC70/metabolismo , Proteínas HSP90 de Choque Térmico/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/fisiología , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Tacrolimus/química , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología
7.
Nucleic Acids Res ; 32(5): 1767-73, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-15026536

RESUMEN

We introduced a series of Pro substitutions within and near the alpha4 helix, a part of the breakage/rejoining region, in human DNA topoisomerase IIalpha, and analyzed if this region is involved in determination of anti-cancer drug sensitivity in a temperature- sensitive yeast strain (top2-4 allele). Among the 19 mutants generated, H759P and N770P showed resistance to etoposide and doxorubicin at the non-permissive temperature, where cell growth depends on activity of the human enzyme. For these residues, mutants with an Ala substitution were further created, in which H759A also showed resistance to etoposide. H759P, H759A and N770P were expressed, purified and subjected to in vitro measurement of drug sensitivity. They generated lower amounts of the etoposide-induced cleavable complexes, and were also found to have lower decatenation activity than the wild-type. In the crystal structure, the yeast equivalent of His759 is found in the vicinity of the Arg713, a putative anchoring residue of the 3'-side of cleaved DNA strands. These results suggest that His759 and the other alpha4 helix residues are involved in the enzymatic activity and drug sensitivity of human DNA topoisomerase IIalpha, via interaction with cleaved DNA.


Asunto(s)
Antineoplásicos/farmacología , ADN-Topoisomerasas de Tipo II/química , ADN-Topoisomerasas de Tipo II/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Aminoácidos/fisiología , Antígenos de Neoplasias , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Etopósido/farmacología , Datos de Secuencia Molecular , Proteínas de Unión a Poli-ADP-Ribosa , Saccharomyces cerevisiae/enzimología , Levaduras/citología , Levaduras/efectos de los fármacos
8.
Artículo en Inglés | MEDLINE | ID: mdl-16838840

RESUMEN

The synthesis and properties of oligonucleotides (ONs) containing 9-(2,3,4-trihydroxybutyl)adenine, A(C2) and A(C3), are described. The ON containing A(C2) involves the 3'-->4' and 3-->5' phosphodiester linkages in the strand, whereas that containing A(C3) possesses the 3'-->4' and 2'-->5' phosphodiester linkages. It was found that incorporation of the analogs, A(C2) or A(C3), into ONs significantly reduces the thermal and thermodynamic stabilities of the ON/DNA duplexes, but does not largely decrease the thermal and thermodynamic stabilities of the ON/RNA duplexes as compared with the case of the ON/DNA duplexes. It was revealed that the base recognition ability of A(C2) is greater than that of A(C3) in the ON/RNA duplexes.


Asunto(s)
Adenina/análogos & derivados , Adenina/química , Emparejamiento Base , Oligonucleótidos/química , Oligonucleótidos/síntesis química , Dicroismo Circular , Calor , Resonancia Magnética Nuclear Biomolecular , Desnaturalización de Ácido Nucleico , Hibridación de Ácido Nucleico , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Estereoisomerismo
9.
Mol Biochem Parasitol ; 143(2): 146-51, 2005 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16005528

RESUMEN

S-adenosylhomocysteine hydrolase is a prospective target for developing new anti-malarial drugs. Inhibition of the hydrolase results in an anti-cellular effect due to the suppression of adenosylmethionine-dependent transmethylations. Based on the crystal structure of Plasmodium falciparum S-adenosylhomocysteine hydrolase which we have determined recently, we performed mutational analyses on P. falciparum and human enzymes. Cys59 and Ala84 of the parasite enzyme, and the equivalent residues on the human enzyme, Thr60 and Gln85, were examined. Mutations of Cys59 and Thr60 caused dramatic impact on inhibition by 2-fluoronoraristeromycin without significant effect both on its kinetic parameters and on inhibition constant against noraristeromycin. In addition, the impact was independent from the electronegativity of the side chain of the substituting residue. These results showed that steric hindrance between a functional group at the 2-position of an adenine nucleoside inhibitor and Thr60 of the human enzyme, not an electrostatic effect, contributed to inhibitor selectivity.


Asunto(s)
Adenosina/análogos & derivados , Adenosilhomocisteinasa/genética , Adenosilhomocisteinasa/metabolismo , Plasmodium falciparum/enzimología , Adenosina/farmacología , Adenosilhomocisteinasa/química , Sustitución de Aminoácidos , Animales , Inhibidores Enzimáticos/farmacología , Humanos , Cinética , Modelos Moleculares , Conformación Molecular , Mutagénesis Sitio-Dirigida , Plasmodium falciparum/genética , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Electricidad Estática
10.
J Mol Biol ; 343(4): 1007-17, 2004 Oct 29.
Artículo en Inglés | MEDLINE | ID: mdl-15476817

RESUMEN

The human malaria parasite Plasmodium falciparum is responsible for the death of more than a million people each year. The emergence of strains of malarial parasite resistant to conventional drug therapy has stimulated searches for antimalarials with novel modes of action. S-Adenosyl-L-homocysteine hydrolase (SAHH) is a regulator of biological methylations. Inhibitors of SAHH affect the methylation status of nucleic acids, proteins, and small molecules. P.falciparum SAHH (PfSAHH) inhibitors are expected to provide a new type of chemotherapeutic agent against malaria. Despite the pressing need to develop selective PfSAHH inhibitors as therapeutic drugs, only the mammalian SAHH structures are currently available. Here, we report the crystal structure of PfSAHH complexed with the reaction product adenosine (Ado). Knowledge of the structure of the Ado complex in combination with a structural comparison with Homo sapiens SAHH (HsSAHH) revealed that a single substitution between the PfSAHH (Cys59) and HsSAHH (Thr60) accounts for the differential interactions with nucleoside inhibitors. To examine roles of the Cys59 in the interactions with nucleoside inhibitors, a mutant PfSAHH was prepared. A replacement of Cys59 by Thr results in mutant PfSAHH, which shows HsSAHH-like nucleoside inhibitor sensitivity. The present structure should provide opportunities to design potent and selective PfSAHH inhibitors.


Asunto(s)
Adenosilhomocisteinasa/química , Plasmodium falciparum/enzimología , Adenosilhomocisteinasa/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Especificidad por Sustrato
11.
J Invest Dermatol ; 133(11): 2514-2521, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23698098

RESUMEN

Generalized pustular psoriasis (GPP) is a rare inflammatory skin disease that can be life-threatening. Recently, it has been reported that familial GPP is caused by homozygous or compound heterozygous mutations of IL36RN. However, the majority of GPP cases are sporadic and it is controversial whether IL36RN mutations are a causative/predisposing factor for sporadic GPP. We searched for IL36RN mutations in two groups of GPP patients in the Japanese population in this study: GPP without psoriasis vulgaris (PV), and GPP with PV. Eleven cases of GPP without PV (GPP alone) and 20 cases of GPP accompanied by PV (GPP with PV) were analyzed. Surprisingly, 9 out of 11 cases of GPP alone had homozygous or compound heterozygous mutations in IL36RN. In contrast, only 2 of 20 cases of GPP with PV had compound heterozygous mutations in IL36RN. The two cases of GPP with PV who had compound heterozygous mutations in IL36RN are siblings, and both cases had PV-susceptible HLA-A*0206. We determined that GPP alone is a distinct subtype of GPP and is etiologically distinguished from GPP with PV, and that the majority of GPP alone is caused by deficiency of the interleukin-36 receptor antagonist due to IL36RN mutations.


Asunto(s)
Pueblo Asiatico/genética , Predisposición Genética a la Enfermedad/genética , Interleucinas/genética , Psoriasis/genética , Psoriasis/patología , Adolescente , Adulto , Anciano , Preescolar , Femenino , Haplotipos , Heterocigoto , Prueba de Histocompatibilidad , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven
16.
Nucleic Acids Symp Ser (Oxf) ; (51): 449-50, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-18029780

RESUMEN

This paper describes the synthesis of 2',5'-oligoadenylate analogs possessing a linker moiety in the place of the second adenosine and their ability to activate human RNase L. Thus, 2-5A analogs (2a-c & 3a-c) possessing -O(CH(2)CH(2))nO- moieties (n = 1 approximately 3) or aromatic ring moieties were prepared. The EC(50) value of the 2-5A analog (2a) incorporating ethylene glycol showed 700 nM in RNase L activation activity.


Asunto(s)
Nucleótidos de Adenina/química , Nucleótidos de Adenina/farmacología , Endorribonucleasas/metabolismo , Oligorribonucleótidos/química , Oligorribonucleótidos/farmacología , Nucleótidos de Adenina/síntesis química , Adenosina/química , Activación Enzimática , Etilenos/química , Humanos , Oligorribonucleótidos/síntesis química
17.
J Org Chem ; 70(20): 7925-35, 2005 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-16277312

RESUMEN

[Chemical reaction: See text] The synthesis and properties of a nucleic acid analogue consisting of a benzene-phosphate backbone are described. The building blocks of the nucleic acid analogue are composed of bis(hydroxymethyl)benzene residues connected to nucleobases via the biaryl-like axis. Stabilities of the duplexes were studied by thermal denaturation. It was found that the thermal stabilities of the duplexes composed of the benzene-phosphate backbone are highly dependent on their sequences. The duplexes with the benzene-phosphate backbone comprised of the mixed sequences were thermally less stable than the natural DNA duplexes, whereas that composed of the homopyrimidine and homopurine sequences was thermally and thermodynamically more stable than the corresponding natural DNA duplex. It was suggested that the analogues more efficiently stabilize the duplexes in a B-form duplex rather than in an A-form duplex. Thus, the duplexes consisting of the benzene-phosphate backbone, especially composed of the homopyrimidine and homopurine sequences, may offer a novel structural motif useful for developing novel materials applicable in the fields of bio- and nanotechnologies.


Asunto(s)
Derivados del Benceno , Ácidos Nucleicos/química , Ácidos Nucleicos/síntesis química , Fosfatos , Secuencia de Bases , Dicroismo Circular , Estabilidad de Medicamentos , Indicadores y Reactivos , Modelos Moleculares , Peso Molecular , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/química , Oligorribonucleótidos/síntesis química , Oligorribonucleótidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Termodinámica
18.
Bioorg Med Chem Lett ; 14(17): 4431-4, 2004 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-15357966

RESUMEN

This paper described synthesis of 2',5'-oligoadenylate (2-5A) analogs containing the purine acyclonucleoside, 9-[(2'S,3'R)-2',3',4'-trihydroxybutyl]adenine (2). The ability of the analogs to activate recombinant human RNase L was evaluated using 5'-32P-r(C11U2C7)-3' as a substrate. The EC50 value (the concentration of the 2-5A required to cleave half of the RNA) of the parent 2-5A tetramer 13 was 1.0 nM, whereas those of the analog 14 incorporating 2 at the second position from the 5'-end and the analog 15 incorporating 2 at the third position from the 5'-end were 9.0 and 1.7 nM, respectively. The analogs 14 and 15 were only 9- and 1.7-fold less potent than the parent 2-5A 13 itself, in RNase L activation ability. Furthermore, the oligodeoxynucleotide containing 2 was more resistant to nucleolytic hydrolysis by snake venom phosphodiesterase (a 3'-exonuclease) than the unmodified oligodeoxynucleotide. Thus, incorporation of an acyclonucleoside into 2-5A may be useful for developing an antiviral agent based on the 2-5A system.


Asunto(s)
Nucleótidos de Adenina/síntesis química , Adenina/síntesis química , Endorribonucleasas/metabolismo , Nucleósidos/síntesis química , Oligorribonucleótidos/síntesis química , Adenina/metabolismo , Nucleótidos de Adenina/metabolismo , Activación Enzimática , Humanos , Nucleósidos/metabolismo , Oligorribonucleótidos/metabolismo , Proteínas Recombinantes/metabolismo
19.
Nucleic Acids Symp Ser (Oxf) ; (48): 17-8, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-17150456

RESUMEN

This paper describes DNA analog synthesis consisting of the benzene-phosphate backbone, of which the monomer units are built from benzene-core units connected via a biaryl-like axis to the nucleobases. It was found that the duplex comprised of the analogs had a non-helical structure and was thermally more stable than the corresponding natural DNA duplex.


Asunto(s)
Benceno/química , ADN/química , ADN/síntesis química , Fosfatos/química , Dicroismo Circular , Cristalografía por Rayos X , ADN/efectos de la radiación , Conformación de Ácido Nucleico/efectos de la radiación , Ácidos Nucleicos Heterodúplex/efectos de la radiación , Compuestos Organofosforados/síntesis química , Compuestos Organofosforados/química , Temperatura de Transición , Rayos Ultravioleta
20.
Biochemistry ; 41(24): 7610-6, 2002 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-12056892

RESUMEN

A number of compounds used for cancer chemotherapy exert their effects by inhibiting DNA replication. New inhibitors of DNA polymerases, therefore, could be potential candidates for new anti-cancer drugs. This study tested the effects of two phenalenone-skeleton-based compounds, which were isolated from a marine-derived fungus Penicillium sp., sculezonone-B (SCUL-B) and sculezonone-A (SCUL-A), upon DNA polymerase activity. Both compounds inhibited bovine DNA polymerases alpha and gamma, moderately affected the activity of DNA polymerase epsilon, and had almost no effect on HIV-reverse transcriptase and an E. coli DNA polymerase I Klenow fragment. Most notably, whereas SCUL-A inhibited pol beta (IC(50) = 17 microM), SCUL-B has only a weak influence upon this polymerase (IC(50) = 90 microM). Kinetic studies showed that inhibition of both DNA polymerases alpha and beta by either SCUL-A or SCUL-B was competitive with respect to dTTP substrate and noncompetitive with the template-primer. Whereas pol alpha inhibition by SCUL-B is competitive with respect to dATP, the inhibition by SCUL-A was found to be a mixed type with dATP substrate. The K(i) values of SCUL-B were calculated to be 1.8 and 6.8 microM for DNA polymerases alpha and gamma, respectively. The K(i) of DNA polymerase beta for SCUL-A was 12 microM and that for DNA polymerase alpha, 16 microM. Therefore, deletion of the OH-group at C12 enhanced inhibition of DNA polymerase beta. Since computational analyses of these two inhibitors revealed a remarkable difference in the distribution of negative electrostatic charge on the surface of molecules, we infer that different electrostatic charges might elicit different inhibition spectra from these two compounds.


Asunto(s)
Bivalvos/microbiología , Inhibidores Enzimáticos/química , Inhibidores de la Síntesis del Ácido Nucleico , Penicillium/química , Fenalenos , Hidrocarburos Policíclicos Aromáticos/química , Compuestos Policíclicos/química , Animales , Bovinos , ADN Polimerasa I/antagonistas & inhibidores , ADN Polimerasa I/metabolismo , ADN Polimerasa II/antagonistas & inhibidores , ADN Polimerasa II/metabolismo , ADN Polimerasa beta/antagonistas & inhibidores , ADN Polimerasa beta/metabolismo , ADN Polimerasa gamma , ADN Polimerasa Dirigida por ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Cinética , Hidrocarburos Policíclicos Aromáticos/farmacología , Compuestos Policíclicos/farmacología , Ratas , Electricidad Estática , Especificidad por Sustrato
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